In cells, intracellular propagation of signals is often achieved through the use of a second messenger molecule. Given the complexity and the number of signaling pathways present in the cell, it is not surprising that the enzymes responsible for generating second messengers are under tight regulation. Autoinhibition is a prevalent mode used in nature to regulate enzyme function. Such is the case for guanylyl cyclases - a class of enzymes responsible for generating the second messenger cGMP from GTP. Guanylyl cyclases (GCs) exist both as transmembrane receptors and as soluble cytoplasmic enzymes. The activity of transmembrane GCs is regulated by an associated protein kinase-like domain and ATP. That of sGCs relies on a heme-binding regulatory region. Despite the involvement of cGMP in many aspects of cell physiology, the regulation of guanylyl cyclases remains poorly understood, partly due to the lack of structural information available on them. This research plan will investigate activity and regulation of guanylyl cyclases from a structural perspective. Using GC-C as a transmembrane cyclase model, we will investigate the allosteric regulation of the enzyme. In particular, the role of the protein kinase-like domain in modulating cyclase activity in response to adenine nucleotides. Constructs suitable for crystallization will be made and biochemically characterized. Similarly, the crystal structure of the regulatory region of sGC bound to different ligands should yield insight into the structural and chemical changes associated with increase in cyclase activity and mode of regulation. Aberrant expression and activity of guanylyl cyclases has been associated with pathophysiological processes like colon cancers, enterotoxin dependent diarrheas, and cardiovascular diseases. A detailed molecular understanding of guanylyl cyclase regulation and function will be useful in the design of novel therapies for these diseases.